Combination of a cdk inhibitor and 5-fu for the tratment of cancer

ABSTRACT

A first aspect of the invention relates to a combination comprising a CDK inhibitor and 5-FU, or a prodrug thereof. A second aspect of the invention relates to a pharmaceutical product comprising a CDK inhibitor and 5-FU, or a prodrug thereof, as a combined preparation for simultaneous, sequential or separate use in therapy. A third aspect of the invention relates to a method of treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a CDK inhibitor and 5-FU, or a prodrug thereof, to a subject.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical combination suitablefor the treatment of cancer and other proliferative disorders.

BACKGROUND TO INVENTION

Initiation, progression, and completion of the mammalian cell cycle areregulated by various cyclin-dependent kinase (CDK) complexes, which arecritical for cell growth. These complexes comprise at least a catalytic(the CDK itself) subunit and a regulatory (cyclin) subunit. Some of themore important complexes for cell cycle regulation include cyclin A(CDK1—also known as cdc2, and CDK2), cyclin B1-B3 (CDK1), cyclin C(CDK8), cyclin D1-D3 (CDK2, CDK4, CDK5, CDK6), cyclin E (CDK2), cyclinsK and T (CDK9) and cyclin H (CDK7). Each of these complexes is involvedin a particular phase of the cell cycle.

The activity of CDKs is regulated post-translationally, by transitoryassociations with other proteins, and by alterations of theirintracellular localisation. Tumour development is closely associatedwith genetic alteration and deregulation of CDKs and their regulators,suggesting that inhibitors of CDKs may be useful anti-cancertherapeutics. Indeed, early results suggest that transformed and normalcells differ in their requirement for e.g. cyclin A/CDK2 and that it maybe possible to develop novel antineoplastic agents devoid of the generalhost toxicity observed with conventional cytotoxic and cytostatic drugs.

The function of CDKs is to phosphorylate and thus activate or deactivatecertain proteins, including, for example, retinoblastoma proteins,lamins, histone H1, and components of the mitotic spindle. The catalyticstep mediated by CDKs involves a phospho-transfer reaction from ATP tothe macromolecular enzyme substrate. Several groups of compounds(reviewed in N. Gray, L. Détivaud, C. Doerig, L. Meijer, Curr. Med.Chem. 1999, 6, 859) have been found to possess anti-proliferativeproperties by virtue of CDK-specific ATP antagonism.

Roscovitine is the compound6-benzylamino-2-[(R)-1-ethyl-2-hydroxyethylamino]-9-isopropylpurine.Roscovitine has been demonstrated to be a potent inhibitor of cyclindependent kinase enzymes, particularly CDK2. This compound is currentlyin development as an anti-cancer agent. CDK inhibitors are understood toblock passage of cells from the G1/S and the G2/M phase of the cellcycle. Roscovitine has also been shown to be an inhibitor ofretinoblastoma phosphorylation and therefore implicated as acting morepotently on Rb positive tumors.

It well established in the art that active pharmaceutical agents canoften be given in combination in order to optimise the treatment regime.The present invention therefore seeks to provide a new combination ofknown pharmaceutical agents that is particularly suitable for thetreatment of proliferative disorders, especially cancer. Morespecifically, the invention centres on the surprising and unexpectedeffects associated with using certain pharmaceutical agents incombination.

STATEMENT OF INVENTION

In a first aspect, the invention provides a combination comprising a CDKinhibitor and 5-FU, or a prodrug thereof.

A second aspect provides a pharmaceutical composition comprising acombination according to the invention admixed with a pharmaceuticallyacceptable carrier, diluent or excipient.

A third aspect relates to the use of a combination according to theinvention in the preparation of a medicament for treating aproliferative disorder.

A fourth aspect relates to a pharmaceutical product comprising a CDKinhibitor and 5-FU, or a prodrug thereof, as a combined preparation forsimultaneous, sequential or separate use in therapy.

A fifth aspect relates to a method of treating a proliferative disorder,said method comprising simultaneously, sequentially or separatelyadministering a CDK inhibitor and 5-FU, or a prodrug thereof, to asubject.

A sixth aspect relates to the use of a CDK inhibitor in the preparationof a medicament for the treatment of a proliferative disorder, whereinsaid treatment comprises simultaneously, sequentially or separatelyadministering a CDK inhibitor and 5-FU, or a prodrug thereof, to asubject.

A seventh aspect relates to the use of a CDK inhibitor and 5-FU, or aprodrug thereof, in the preparation of a medicament for treating aproliferative disorder.

An eighth aspect relates to the use of a CDK inhibitor in thepreparation of a medicament for the treatment of a proliferativedisorder, wherein said medicament is for use in combination therapy with5-FU, or a prodrug thereof.

A ninth aspect relates to the use of 5-FU, or a prodrug thereof, in thepreparation of a medicament for the treatment of a proliferativedisorder, wherein said medicament is for use in combination therapy witha CDK inhibitor.

DETAILED DESCRIPTION

The preferred embodiments as set out below are applicable to all theabove-mentioned aspects of the invention.

As mentioned above, the present invention relates to a combinationcomprising a CDK inhibitor and 5-FU, or a prodrug thereof.5-Fluorouracil (5-FU) is an antitumour antimetabolite, extensively usedin chemotherapeutic regimes against solid tumours, particularly those ofgastric or colonic origin. 5-Fluorouracil was developed in 1957 based onthe observation that tumour cells utilized the base pair uracil for DNAsynthesis more efficiently than normal cells of the intestinal mucosa.5-FU is a fluorinated pyrimidine that is metabolized intracellularly toits active form, fluorodeoxyuridine monophophate (FdUMP). The activeform inhibits DNA synthesis by inhibiting the normal production ofthymidine. 5-FU is cell cycle phase-specific (S-phase), i.e. it blockscell progression from the S-phase of the cell cycle.

The effect of drug combinations is inherently unpredictable and there isoften a propensity for one drug to partially or completely inhibit theeffects of the other. The present invention is based on the surprisingobservation that administering 5-FU and a CDK inhibitor (for example,roscovitine) in combination, either simultaneously, separately orsequentially, does not lead to any adverse interaction between the twoagents. The unexpected absence of any such antagonistic interaction iscritical for clinical applications.

Preferably, the combination has a synergistic effect, i.e. thecombination is synergistic.

As mentioned above, one aspect of the invention relates to apharmaceutical product comprising a CDK inhibitor and 5-FU, or a prodrugthereof, as a combined preparation for simultaneous, sequential orseparate use in therapy.

The CDK inhibitor and 5-FU, or prodrug thereof, may be administeredsimultaneously, in combination, sequentially or separately (as part of adosing regime).

As used herein, “simultaneously” is used to mean that the two agents areadministered concurrently, whereas the term “in combination” is used tomean they are administered, if not simultaneously, then “sequentially”within a timeframe that they both are available to act therapeuticallywithin the same time-frame. Thus, administration “sequentially” maypermit one agent to be administered within 5 minutes, 10 minutes or amatter of hours after the other provided the circulatory half-life ofthe first administered agent is such that they are both concurrentlypresent in therapeutically effective amounts. The time delay betweenadministration of the components will vary depending on the exact natureof the components, the interaction therebetween, and their respectivehalf-lives.

In contrast to “in combination” or “sequentially”, “separately” is usedherein to mean that the gap between administering one agent and theother is significant i.e. the first administered agent may no longer bepresent in the bloodstream in a therapeutically effective amount whenthe second agent is administered.

One aspect of the present invention relates to the use of a CDKinhibitor in the preparation of a medicament for the treatment of aproliferative disorder, wherein said treatment comprises administeringto a subject simultaneously, sequentially or separately 5-FU, or aprodrug thereof, and a CDK inhibitor.

Preferably, the CDK inhibitor and 5-FU, or prodrug thereof, areadministered simultaneously or sequentially.

In one preferred embodiment, the 5-FU, or prodrug thereof, and CDKinhibitor are administered simultaneously.

In one particularly preferred embodiment, the CDK inhibitor isadministered to the subject prior to sequentially or separatelyadministering 5-FU, or prodrug thereof, to said subject.

Another aspect of the invention relates to a method of treating aproliferative disorder comprising the sequential administration of atherapeutically effective amount of CDK inhibitor followed by atherapeutically effective amount of 5-FU.

Another aspect of the invention relates to the use of roscovitine in themanufacture of a medicament for use in the treatment of proliferativedisorders comprising the sequential administration of a therapeuticallyeffective amount of CDK inhibitor followed by a therapeuticallyeffective amount of 5-FU.

In an alternative preferred embodiment, 5-FU, or prodrug thereof, isadministered to the subject prior to sequentially or separatelyadministering a CDK inhibitor to said subject.

In one particularly preferred embodiment, the CDK inhibitor and 5-FU, orprodrug thereof, are administered sequentially.

In one preferred embodiment of the invention, the CDK inhibitor and5-FU, or prodrug thereof, are each administered in a therapeuticallyeffective amount with respect to the individual components.

In another preferred embodiment of the invention, the CDK inhibitor and5-FU, or a prodrug thereof, are each administered in a subtherapeuticamount with respect to the individual components.

Another aspect of the invention relates to the use of a CDK inhibitorand 5-FU, or a prodrug thereof, in the preparation of a medicament fortreating a proliferative disorder.

Yet another aspect of the invention relates to the use of a CDKinhibitor in the preparation of a medicament for the treatment of aproliferative disorder, wherein said medicament is for use incombination therapy with 5-FU, or a prodrug thereof.

A further aspect of the invention relates to the use of 5-FU, or aprodrug thereof, in the preparation of a medicament for the treatment ofa proliferative disorder, wherein said medicament is for use incombination therapy with a CDK inhibitor.

As used herein, the term “combination therapy” refers to therapy inwhich the 5-FU, or prodrug thereof, and CDK inhibitor are administered,if not simultaneously, then sequentially within a timeframe that theyboth are available to act therapeutically within the same time-frame.

As used herein the phrase “preparation of a medicament” includes the useof the components of the invention directly as the medicament inaddition to their use in any stage of the preparation of such amedicament.

The term “proliferative disorder” is used herein in a broad sense toinclude any disorder that requires control of the cell cycle, forexample cardiovascular disorders such as restenosis and cardiomyopathy,auto-immune disorders such as glomerulonephritis and rheumatoidarthritis, dermatological disorders such as psoriasis,anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria,emphysema and alopecia. In these disorders, the components of thepresent invention may induce apoptosis or maintain stasis within thedesired cells as required.

Preferably, the proliferative disorder is a cancer or leukaemia, mostpreferably cancer.

In a more preferred embodiment, the proliferative disorder is a softtissue cancer, for example, breast cancer.

In another preferred embodiment, the proliferative disorder iscolorectal cancer, head or neck cancer, cervical or pancreatic cancer.

In a particularly preferred embodiment, the invention relates to the useof the combination described herein in the treatment of a CDK dependentor sensitive disorder. CDK dependent disorders are associated with anabove normal level of activity of one or more CDK enzymes. Suchdisorders preferably associated with an abnormal level of activity ofCDK2 and/or CDK4. A CDK sensitive disorder is a disorder in which anaberration in the CDK level is not the primary cause, but is downstreamof the primary metabolic aberration. In such scenarios, CDK2 and/or CDK4can be said to be part of the sensitive metabolic pathway and CDKinhibitors may therefore be active in treating such disorders. Suchdisorders are preferably cancer or leukaemic disorders.

Preferably, the CDK inhibitor is an inhibitor of CDK2 and/or CDK4. Morepreferably the CDK inhibitor is selected from roscovitine, purvalanol A,purvalanol B, olomucine and other 2,6,9-trisubstituted purines asdescribed in WO97/20842, WO98/05335 (CV Therapeutics), WO99/07705(Regents of the University of California).

Even more preferably the CDK inhibitor is selected from roscovitine andpurvalanol A.

In one particularly preferred embodiment, the CDK inhibitor isroscovitine.

Roscovitine is the compound2-[(1-ethyl-2-hydroxyethyl)amino]-6-benzylamine-9-isopropylpurine, alsodescribed as2-(1-D,L-hydroxymethylpropylamino)-6-benzylamine-9-isopropylpurine. Asused herein, the term “roscovitine” encompasses the resolved R and Senantiomers, mixtures thereof, and the racemate thereof.

Many anti-cancer agents are given in combination in order to optimisethe treatment regime. 5-FU has been used in combination withcyclophosphamide, methotrexate (Int J Cancer (1981) 28, 91-96) andtogether with leucovorin in a dosing regime with cisplatin (Jpn J ClinOne (2001) 31, 605-609). This latter disclosure provides summary of 5-FUcombinations that have been attempted with a view to improving treatmentof advance gastric cancer. Bible K C and Kaufmann S H, Cancer Res.(1997) 57: 3375-3380, described the sequential administration offlavopurinol and 5-FU. However, to date there has been no suggestion ofadministering 5-FU, or a prodrug thereof, in combination withroscovitine.

Even more preferably, the combination is a synergistic combinationcomprising roscovitine and 5-FU, or a prodrug thereof.

In a preferred embodiment, the combination of 5-FU, or a prodrugthereof, and roscovitine produces an enhanced effect as compared toeither drug administered alone. The surprising nature of thisobservation is in contrast to that expected on the basis of the priorart.

In one particularly preferred embodiment of the invention, the prodrugis capecitabine. Thus, preferably, the combination comprises roscovitineand capecitabine.

Pharmaceutical Compositions

Although the components of the present invention (including theirpharmaceutically acceptable salts, esters and pharmaceuticallyacceptable solvates) can be administered alone, for human therapy theywill generally be administered in admixture with a pharmaceuticalcarrier, excipient or diluent.

A preferred embodiment of the invention therefore relates to apharmaceutical composition comprising a CDK inhibitor and 5-FU, or aprodrug thereof, admixed with a pharmaceutically acceptable excipient,diluent or carrier.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients, 2^(nd) Edition, (1994), Editedby A Wade and P J Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like.

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Salts/Esters

The agents of the present invention can be present as salts or esters,in particular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the agents of the invention includesuitable acid addition or base salts thereof. A review of suitablepharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19(1977). Salts are formed, for example with strong inorganic acids suchas mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalicacids; with strong organic carboxylic acids, such as alkanecarboxylicacids of 1 to 4 carbon atoms which are unsubstituted or substituted(e.g., by halogen), such as acetic acid; with saturated or unsaturateddicarboxylic acids, for example oxalic, malonic, succinic, maleic,fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, forexample ascorbic, glycolic, lactic, malic, tartaric or citric acid; withaminoacids, for example aspartic or glutamic acid; with benzoic acid; orwith organic sulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonicacids which are unsubstituted or substituted (for example, by a halogen)such as methane- or p-toluene sulfonic acid.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified. Organic acidsinclude carboxylic acids, such as alkanecarboxylic acids of 1 to 12carbon atoms which are unsubstituted or substituted (e.g., by halogen),such as acetic acid; with saturated or unsaturated dicarboxylic acid,for example oxalic, malonic, succinic, maleic, fumaric, phthalic ortetraphthalic; with hydroxycarboxylic acids, for example ascorbic,glycolic, lactic, malic, tartaric or citric acid; with aminoacids, forexample aspartic or glutamic acid; with benzoic acid; or with organicsulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonic acids which areunsubstituted or substituted (for example, by a halogen) such asmethane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoholsof 1-12 carbon atoms which may be unsubstituted or substituted, e.g. bya halogen).

Enantiomers/Tautomers

The invention also includes where appropriate all enantiomers andtautomers of the agents. The man skilled in the art will recognisecompounds that possess optical properties (one or more chiral carbonatoms) or tautomeric characteristics. The corresponding enantiomersand/or tautomers may be isolated/prepared by methods known in the art.

Stereo and Geometric Isomers

Some of the agents of the invention may exist as stereoisomers and/orgeometric isomers, e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms. The present invention contemplates the use of all theindividual stereoisomers and geometric isomers of those agents, andmixtures thereof. The terms used in the claims encompass these forms,provided said forms retain the appropriate functional activity (thoughnot necessarily to the same degree).

The present invention also includes all suitable isotopic variations ofthe agents or pharmaceutically acceptable salts thereof. An isotopicvariation of an agent of the present invention or a pharmaceuticallyacceptable salt thereof is defined as one in which at least one atom isreplaced by an atom having the same atomic number but an atomic massdifferent from the atomic mass usually found in nature. Examples ofisotopes that can be incorporated into the agent and pharmaceuticallyacceptable salts thereof include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Certainisotopic variations of the agent and pharmaceutically acceptable saltsthereof, for example, those in which a radioactive isotope such as ³H or¹⁴C is incorporated, are useful in drug and/or substrate tissuedistribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with isotopes such as deuterium,i.e., ²H, may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example, increased in vivo half-life orreduced dosage requirements and hence may be preferred in somecircumstances. Isotopic variations of the agents of the presentinvention and pharmaceutically acceptable salts thereof can generally beprepared by conventional procedures using appropriate isotopicvariations of suitable reagents.

Solvates

The present invention also includes solvate forms of the agents of thepresent invention. The terms used in the claims encompass these forms.

Polymorphs

The invention furthermore relates to agents of the present invention intheir various crystalline forms, polymorphic forms and (an)hydrousforms. It is well established within the pharmaceutical industry thatchemical compounds may be isolated in any of such forms by slightlyvarying the method of purification and or isolation form the solventsused in the synthetic preparation of such compounds.

Chemical Derivatives

The invention also relates to combinations which comprise derivatives ofthe agents. The term “derivative” as used herein includes chemicalmodification of an agent. Illustrative of such chemical modificationswould be replacement of hydrogen by a halo group, an alkyl group, anacyl group or an amino group.

Prodrugs

The invention further includes agents of the present invention inprodrug form. Such prodrugs are generally compounds wherein one or moreappropriate groups have been modified such that the modification may bereversed upon administration to a human or mammalian subject. Suchreversion is usually performed by an enzyme naturally present in suchsubject, though it is possible for a second agent to be administeredtogether with such a prodrug in order to perform the reversion in vivo.Examples of such modifications include esters (for example, any of thosedescribed above), wherein the reversion may be carried out be anesterase etc. Other such systems will be well known to those skilled inthe art.

In one particularly preferred embodiment, the 5-FU is in the form of aprodrug, preferably capecitabine. Capecitabine is a 5-FU prodrug whichis particularly suitable for oral administration.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal,intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal,intravenous, nasal, buccal or sublingual routes of administration.

For oral administration, particular use is made of compressed tablets,pills, tablets, gellules, drops, and capsules. Preferably, thesecompositions contain from 1 to 2000 mg and more preferably from 50-1000mg, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Thepharmaceutical compositions of the present invention may also be in formof suppositories, pessaries, suspensions, emulsions, lotions, ointments,creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredients can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredients can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Injectable forms may contain between 10-1000 mg, preferably between10-500 mg, of active ingredient per dose.

Compositions may be formulated in unit dosage form, i.e., in the form ofdiscrete portions containing a unit dose, or a multiple or sub-unit of aunit dose.

In a particularly preferred embodiment, the combination orpharmaceutical composition of the invention is administeredintravenously.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific agents employed, the metabolic stabilityand length of action of that agent, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Depending upon the need, the agent may be administered at a dose of from0.1 to 30 mg/kg body weight, or from 2 to 20 mg/kg, more preferably from0.1 to 1 mg/kg body weight.

As described above, each active component, the CDK inhibitor and 5-FU,or a prodrug thereof, are administered in a therapeutically effectiveamount preferably in the form of a pharmaceutically acceptablecomposition. These amounts will be familiar to those skilled in the art.By way of guidance, 5-FU is typically administered intravenously, orallyor topically. Intravenous and oral doses typically comprise 250 mg or500 mg 5-FU and are administered in accordance to a physicians directionat a total dosage depending on the weight of a patient e.g. orally at 15mg/kg weekly, maximum dose 1 g/day, or intravenously 12 mg/kg over 4hours, or 24-49 mg/kg over 24 hours daily for 5 days. Oral dosages aretypically administered in capsules, whereas intra-venous administrationis generally administered over a number of hours, typically 4 hours.

Preferably, roscovitine is administered as an orally or intravenously ata dosage of from 1 to 5 g/day. 5-FU is then administered in the mannerdeemed most suitable at an appropriate dosage as discussed above.Preferably, the 5-FU is administered at least 24 hours after theadministration of roscovitine.

Roscovitine is typically administered orally or intravenously at adosage of from about 0.05 to about 5 g/day, preferably from about 0.5 toabout 5 g/day or 1 to about 5 g/day, and even more preferably from about1 to about 3 g/day. Alternatively, roscovitine is preferablyadministered at a dosage of about 0.4 to about 3 g/day. Roscovitine ispreferably administered orally in tablets or capsules. The total dailydose of roscovitine can be administered as a single dose or divided intoseparate dosages administered two, three or four time a day.

The present invention is further described by way of example and withreference to the following figures wherein:

FIG. 1 shows the effect of treatment with roscovitine (referred toherein as “CYC202” or “202”), 5-FU, and [roscovitine+5-FU] on the cellcycle distribution of HT29 cells.

FIG. 2 shows the effect of sequential treatment with roscovitine and5-FU on the cell cycle distribution of HT29 cells.

FIG. 3 shows the effect of treatment with roscovitine, 5-FU and[roscovitine+5-FU] on the activation of caspase-3 in HT29 cells.

FIG. 4 shows the effect of sequential treatment with roscovitine and5-FU on the activation of caspase-3 in HT29 cells.

FIG. 5 shows the effect of combined treatment with roscovitine and 5-FUon the proliferation of HT29 cells, as determined by the incorporationof BrdU.

FIG. 6 shows the effect of 5-FU pretreatment on MCF7 cells; cell number(as % of control) versus drug dose (as % of IC₅₀).

FIG. 7 shows the effect of roscovitine pretreatment on MCF7 cells; cellnumber (as % of control) versus drug dose (as % of IC₅₀).

FIG. 8 shows the effect of concurrent roscovitine/5-FU treatment on MCF7cells; cell number (as % of control) versus drug dose (as % of IC₅₀).

FIG. 9 shows the in vitro anticancer efficacy of roscovitine incombination with 5-fluorouracil (5-FU) in the human mammary cancerxenograft MAXF 857.

EXAMPLES

Materials and Methods

HT29 human colorectal carcinoma cells were treated with roscovitineand/or 5-FU. The compounds were given singly, concomitantly and insequence. The effects of the sequence of administration of roscovitinewith 5-FU were analysed by flow cytometry and by assaying caspase-3levels (an early marker of induction of apoptosis.)

Cell Culture

The human colorectal cancer cell line HT29 was obtained from theEuropean Collection of Animal Cell Cultures. Cells were grown inDulbecco's Modified Eagle's medium supplemented with 10% v/v fetal calfserum (Perbio), 100 U/ml penicillin and 100 μg/ml streptomycin. Cellswere grown at 37° C., 5% v/v CO₂ in a humidified atmosphere andharvested using 0.05% w/v trypsin, 0.02% w/v EDTA. Cells were washed inmedia to inactivate trypsin before reseeding or analysis.

Drug Treatment

Drug treatment concentrations were selected on the basis of IC₅₀ valuescalculated by a cellular cytotoxicity assay. Roscovitine was dosed ateither IC₅₀ (20 μM) or 2×IC₅₀ (40 μM) and 5-FU at IC₅₀ (1 μM) or0.5×IC₅₀ (0.5 μM). For combined and sequential treatments all possiblecombinations of drug and concentration were evaluated.

Analysis of Cell Cycle Distribution

HT29 cells were seeded onto 90 mm diameter plates at 1×10⁶ cells perplate and incubated for 24 hours. Cells were treated with eitherroscovitine, 5-FU or roscovitine+5-FU at the relevant concentrations for48 hours, except where sequential drug treatment was to be applied. Insamples where sequential application of the drugs was to be evaluated,after 24 hours exposure to the first drug, media was removed from theplates to tubes and the relevant second drug added. After mixing, mediawas returned to the relevant plates, which were incubated for a further24 hours. Both detached and adherent cells were then harvested. Afterwashing once in PBS, cells were fixed in ice cold 70% v/v ethanol inwater and stored at −20° C. Cells were washed twice in PBS+1% w/v BSA toremove fixative and re-suspended in PBS containing 0.1% v/v triton-X, 50μg/ml propidium iodide and 50 μg/ml RNaseA. After incubation at roomtemperature for 20 minutes, cells were analysed using flow cytometry.

Activated Caspase-3 Assay

HT29 cells were seeded, treated with drug and harvested in the same wayas for the determination of cell cycle distribution. Cells were fixed in1% w/v paraformaldehyde for 30 minutes at 37° C., washed once in PBS,re-suspended in ice cold 70% v/v ethanol and stored at −20° C. beforeanalysis. Cells were washed twice in PBS+1% w/v BSA and re-suspended in120 μl of FITC conjugated anti-activated caspase-3 antibody (Pharmingen)diluted 1:5 in PBS+1% BSA, and incubated at room temperature for 30minutes protected from light. After washing once on PBS+1% BSA, cellswere re-suspended in PBS containing 0.1% v/v triton-X, 50 μg/mlpropidium iodide and 50 μg/ml RNaseA. After incubation at roomtemperature protected from light for 20 minutes, samples were analysedby flow cytometry.

Assessment of Cell Proliferation by Incorporation of BrdU

HT29 cells were seeded and treated with drug in the same way as for thedetermination of cell cycle distribution. After 24 hours, media wasreplaced with that containing 10 μM BrdU and incubated for 30 minutes.Detached cells were harvested, plates were washed twice in PBS andadherent cells harvested by trypsinisation. Adherent and detached cellswere pooled, washed in PBS, fixed in ice cold 70% v/v ethanol and storedat 4° C. Cells were washed twice in PBS+1% BSA and treated with 2M HClfor 20 minutes, followed by a further three washes in PBS. Cells werepelleted and 2 μl of anti-BrdU antibody (Pharmingen) added to thepellet. Cells were left at room temperature for 30 minutes and washedonce in PBS+1% BSA. 50 μl of FITC conjugated anti-mouse F(ab′)₂ (Dako)diluted 1:10 in PBS+1% BSA was added to pelleted cells and samplesincubated for 30 minutes at room temperature protected from light. Afterwashing once on PBS+1% BSA, cells were re-suspended in PBS containing0.1% v/v triton-X, 50 μg/ml propidium iodide and 50 μg/ml RNaseA. Afterincubation at room temperature protected from light for 20 minutes,samples were analysed by flow cytometry.

Flow Cytometry

A Becton Dickinson LSR flow cytometer was used for these studies, inaccordance with the manufacturers recommendations. The argon ion laserset at 488 nm was used as an excitation source. Where used, activatedcaspase-3 and BrdU positive cells were designated as such on the basisof green fluorescence (530±28 nm), acquired on a logarithmic scale. Redfluorescence (575±26 nm) was acquired on a linear scale and pulse widthanalysis was used to exclude cell doublets and aggregates from theanalysis. Cells with a DNA content of between 2n and 4n were designatedas being in G1, S or G2/M phases of the cell cycle, as defined by thelevel of red fluorescence. Cells showing less than 2n DNA content weredesignated as sub-G1 cells. The number of cells in each cell cyclecompartnent was expressed as a percentage of the total number of cellspresent.

Results

Cell Cycle Analysis

The effect of treatment with roscovitine, 5-FU and roscovitine+5-FU onthe cell cycle distribution of HT29 cells is shown in FIG. 1.

Treatment with roscovitine at both 20 μM and 40 μM causes accumulationof cells in G2/M, and treatment with 0.5 μM and 1 μM 5-FU causesaccumulation of cells in S phase, both in a dose dependent manner. Whencells were treated concurrently with 20 μM roscovitine and either 0.5 μMor 1 μM 5-FU, accumulations in S phase were seen indicating that, atboth concentrations, 5-FU can prevent the G2/M block of 20 μMroscovitine. However, when the level of roscovitine was increased to 40μM, accumulations in G2/M were seen at both 0.5 μM and 1 μM 5-FU,indicating that by increasing the level of roscovitine, the effect of5-FU can be overcome.

The effect of sequential treatment with roscovitine and 5-FU on the cellcycle distribution of HT29 cells is shown in FIG. 2.

When roscovitine is dosed prior to 5-FU, the cell cycle distributionsare much the same as those obtained for roscovitine alone. Increasingthe concentration of 5-FU from 0.5 μM to 1 μM had no obvious effect oncell cycle distribution. When 5-FU was dosed prior to roscovitine theaccumulations in G2/M, usually seen on treatment with roscovitine werenot as marked, particularly in those samples treated with 40 μMroscovitine. In samples treated with 1 μM 5-FU followed by 40 μMroscovitine there was a marked increase in S phase cells. These resultssuggest that 5-FU is capable of blocking the cell cycle effect ofroscovitine.

Caspase Activation

FIG. 3 shows the effect of treatment with roscovitine, 5-FU and(roscovitine+5-FU) on the activation of caspase-3 in HT29 cells.

Roscovitine induces activation of caspase-3 (an early marker ofapoptotic cell death) in a dose dependent manner, as does 5-FU, albeitto a lesser extent. When dosed in combination the results are similar tothose seen with roscovitine alone, indicating that concurrent treatmentwith the two drugs at the levels tested does not increase cell death(ie. there is no evidence of an additive or synergistic effect on thebasis of this particular assay).

The effect of sequential treatment with roscovitine and 5-FU on theactivation of caspase-3 in HT29 cells is shown in FIG. 4.

Treatment with roscovitine prior to 5-FU showed high levels of caspase-3activation (cell death), with similar levels being seen in all drugconcentration combinations. More specifically, the administration ofroscovitine prior to 5-FU results in an improved activation ofcaspase-3, and this activation is greater than that achieved by 5-FU orroscovitine alone. Thus, on the basis of this assay the sequentialadministration of a CDK inhibitor, such as roscovitine, followed by 5-FUproduces a maximal effect on the induction of apoptosis as compared toeither drug administered alone. Without wishing to be bound by theory,it is believed that sequential treatment with roscovitine followed by5-FU prevents the 5-FU-induced block in the S-phase.

When 5-FU was dosed prior to roscovitine, the levels of cell death weregreatly reduced suggesting that 5-FU may block the induction of celldeath by roscovitine, when measured by this particular assay.

BrdU Analysis

Treatment with 40 μM roscovitine results in reduced proliferation asshown by the reduction in the number of cells incorporating BrdU (3.8%).Treatment with 1 μM 5-FU, increased the number of cells incorporatingBrdU to 64.3% but the intensity of BrdU labelling was lower than thatseen in the control, which is indicative of a drug which causes arrestin S phase. When dosed concurrently, 40 μM roscovitine+1 μM 5-FUresulted in more cells incorporating BrdU than roscovitine alone. Thisindicates that the presence of 5-FU is blocking the effect ofroscovitine, as suggested by the cell cycle distribution results.Sequential treatment with 40 μM roscovitine prior to 1 μM 5-FU gaveresults similar to those for 40 μM roscovitine alone. Sequentialtreatment with 1 μM 5-FU followed by 40 μM roscovitine showed areduction in the proliferation compared to 5-FU alone and an increasecompared to roscovitine alone, indicating that dosing 5-FU prior toroscovitine may be inhibiting the effect of roscovitine.

The effect of combined treatment with roscovitine and 5-FU on theproliferation of HT29 cells, as determined by the incorporation of BrdUis shown in FIG. 5.

Roscovitine and 5FU Treatment of MCF7 Cells

MCF7 cells were seeded at 3,000/well in 96 well plates. Cell wereallowed to settle overnight, and medium replaced with drug-containingmedium the next day. Nine different drug treatments were tested, intriplicate, in each plate. The IC₅₀ values for the two drugs of interestin MCF7 cells was 8 μM (5-FU) and 10 μM roscovitine. The experimentmodel ranged from treating cells with 100% of the IC₅₀ for 5-FU(treatment 1), to 100% of the IC₅₀ for roscovitine (treatment 7).Treatments 2-6 involved treating cells with a range of different ratiosof 5-FU to roscovitine, as shown in Table 1 below. Two additionalcontrols were also run, which involved treatment with no drug (treatment8) or with 100% IC₅₀ of both drugs (treatment 9). TABLE 1 Treatment Drug1 2 3 4 5 6 7 8 9 5-FU 100 75 60 50 40 25 0 0 100 roscovitine 0 25 40 5060 75 100 0 100

Three different combination strategies were tested:

-   (A) 5-FU pretreatment wherein cells were treated with 5-FU for 24    hours, then the medium was replaced with roscovitine-containing    medium for 48 hours;-   (B) Roscovitine pretreatment wherein cells were treated with with    roscovitine for 24 hours, then 5-FU for 48 hours;-   (C) Cells treated with 5-FU and roscovitine concurrently for 72    hours.

In all three combination strategies, after the 72 hour experimentalperiod, the cells numbers in each well were estimated using the WST1assay (Roche Applied Science Assay Catalogue No. 1 644 807). Within eachplate, cell numbers for each treatment were expressed as a percentage ofcells in the control wells (no drug).

Statistical Analysis of Combination Studies

To interpret the combination curves, statistical comparisons were madewith each test combination (75:25 roscovitine/5-FU) and the endpoints(100:0-roscovitine and 0:100-5-FU). A statistically significantobservation requires that a difference exists between the combination(roscovitine and 5-FU) absorbance value and both endpoint values(roscovitine and 5-FU alone) [Greco et al, The search for synergy; Acritical review from a response surface perspective. Pharmacol; Review47:331-385, 1995; Laska et al, Simple designs and model-free tests forsynergy; Biometrics 50:834-841, 1994]. If the majority of (≧3 of 5) ofthe values are statistically above or below the line (endpoints) thenantagonism or synergy is described, respectively. Otherwise, the patternis more consistent with an additive interaction.

Results

The results for combination strategies (A), (B) and (C) are shown belowin Tables 2, 3 & 4, and FIGS. 6, 7 & 8. TABLE 2 5-FU/Roscovitine Treat-st. ment Run 1 Run 2 Run 3 Run 4 Run 5 average dev 1 31.89 35.51 39.1531.68 41.56 35.96 4.38 2 34.49 26.94 35.7 33.7 39.92 34.15 4.69 3 31.1321.52 31.87 31.95 36.02 30.50 5.37 4 32.43 23.52 29.27 32.87 36.62 30.944.90 5 23.75 20.57 29.73 42.36 35.2 30.32 8.77 6 31.13 18.95 29.14 32.9742.37 30.91 8.39 7 61.93 32.94 41.29 43.09 52.85 46.42 11.19

TABLE 3 5-FU + Roscovitine Treat- st. ment Run 1 Run 2 Run 3 Run 4 Run 5average dev 1 30.7 19.55 30.95 19.64 41.49 28.47 9.19 2 27.68 14.6121.41 14.76 31.68 22.03 7.64 3 23.93 13.65 15.74 11.98 23.56 17.77 5.614 26.43 6.88 13.58 9.69 19.98 15.31 7.92 5 29.24 3.77 13.93 9.1 20.4215.29 9.92 6 32.36 7.75 12.53 9.45 15.96 15.61 9.87 7 22.37 14.29 19.9516.62 30.71 20.79 6.35

TABLE 4 Roscovitine/5-FU Treat- st. ment Run 1 Run 2 Run 3 Run 4 Run 5average dev 1 57.29 40.21 53.15 56.82 36.08 48.71 9.89 2 62.56 44.8243.61 51.48 31.91 46.88 11.25 3 56.78 41.28 38.04 43.52 30.57 42.04 9.594 57.29 37.25 32.61 50.8 29.57 41.50 11.99 5 49.75 38.97 34.76 54.1931.7 41.87 9.70 6 41.58 36.09 35.89 52.07 43.05 41.74 6.61 7 17.84 22.6731.03 40.14 45.76 31.49 11.65

TABLE 5 5-FU/ 5-FU/ Roscovitine/ Roscovitine Roscovitine Roscovitine5-FU 5-FU + Roscovitine 5-FU (%) (%) 100/0  35.96 48.71 28.47 100 075/25 34.15 46.88 22.03 75 25 60/40 30.5 42.04 17.77 60 40 50/50 30.9441.5 15.31 50 50 40/60 30.32 41.87 15.29 40 60 25/75 30.91 41.74 15.6125 75  0/100 46.42 31.49 20.79 0 100Combination Strategy (A)

The results for pretreatment with 5-FU (shown in Tables 2 & 5, FIG. 6)are indicative of a synergistic interaction between 5-FU androscovitine.

Combination Strategy (B)

The results for pretreatment with roscovitine (shown in Tables 3 & 5,FIG. 7) are indicative of an additive interaction between 5-FU androscovitine.

Combination Strategy (C)

The results for concurrent treatment with 5-FU and roscivitine (shown inTables 3 & 5, FIG. 8) are indicative of a synergistic interactionbetween 5-FU and roscovitine.

Clonogenic Assay

Preparation of Single Cell Suspensions From Human Tumor Xenografts

Solid human tumor xenografts growing subcutaneously in serial passagesin thymus aplastic nude mice (NMRI, Naval Medical Research Institute,USA, nu/nu strain, obtained from breeding facility) were removed understerile conditions, mechanically disaggregated and subsequentlyincubated with an enzyme cocktail consisting of collagenase (41 U/ml,Sigma), DNAse I (125 U/ml, Roche), hyaluronidase (100 U/ml, Sigma) anddispase II (1.0 U/ml, Roche) in RPMI 1640-Medium (Life Technologies) at37° C. for 40 minutes. Cells were passed through sieves of 200 μm and 50μm mesh size and washed twice with sterile PBS-buffer (LifeTechnologies). The percentage of viable cells was determined in aNeubauer-hemocytometer using trypan blue exclusion.

Culture Methods

The clonogenic assay was performed in a 24-well format according to amodified two-layer soft agar assay introduced by Hamburger et al[Hamburger, A. W. & S. E. Salmon. 1977; Primary bioassay of human tumorstem cells. Science 197: 461-463]. The bottom layer consisted of 0.2ml/well of Iscove's Modified Dulbecco's Medium (supplemented with 20%(v/v) fetal calf serum and 0.01% (v/v) gentamicin) and 0.75% (w/v) agar.5·10⁴ cells were added to 0.2 ml of the same culture medium supplementedwith 0.4% (w/v) agar and plated in 24-multiwell dishes onto the bottomlayer. Cytostatic drugs were applied by continuous exposure (drugoverlay) in 0.2 ml culture medium 24 hours after seeding in the cells.Every dish included six control wells and drug-treated groups intriplicate at 6 concentrations. In combination studies, roscovitine andthe standard cytotoxic agent were applied simultaneously, 3-foldconcentrated to end up in the respective test concentration which isreached by diffusion through the assay layers. The cytotoxic agent wastested in 6 dilutions and roscovitine was added in 2 constantconcentrations to the respective dilutions. Cultures were incubated at37° C. and 7.5% CO₂ in a humidified atmosphere for 8-20 days andmonitored closely for colony growth using an inverted microscope. Withinthis period, in vitro tumor growth led to the formation of colonies witha diameter of >50 μm. At the time of maximum colony formation, countswere performed with an automatic image analysis system (OMNICON FAS IV,Biosys GmbH). 24 hours prior to evaluation, vital colonies were stainedwith a sterile aqueous solution of2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1mg/ml, 100 μl/well) (Alley, M. C., Uhi, C. B. & M. M. Lieber, 1982.Improved detection of drug cytotoxicity in the soft agar colonyformation assay through use of a metabolizable tetrazolium salt. LifeSci. 31: 3071-3078).

An assay was considered fully evaluable, if the following qualitycontrol criteria were fulfilled:

-   -   Mean number of colonies in the control group wells of        24-multiwell plates≧20 colonies with a colony diameter of >50        μm;    -   Coefficient of variation in the control group≦50%;    -   The positive reference compound 5-fluorouracil (5-FU) (at the        toxic dose of 1000 μg/ml) must effect a colony survival of <20%        of the controls; or    -   Initial plate counts on day 0 or 2<20% of the final control        group count.        Data Evaluation

Drug effects were expressed in terms of the percentage of survival,obtained by comparison of the mean number of colonies in the treatedplates with the mean colony count of the untreated controls (relativecolony count expressed by the test-versus-control-group value, T/C-value[%]):$\frac{T}{C} = {\frac{{colony}\quad{count}_{{treated}\quad{group}}}{{colony}\quad{count}_{{control}\quad{group}}} \cdot {{100\quad\left\lbrack \% \right\}}.}}$

In combination studies, T/C-values obtained for the combination at eachdrug dosage were compared to the T/C-values obtained with roscivitine orthe standard agent at the respective concentration alone. A benefit ofcombination was obtained when T/C-values from combinations weresignificantly lower than the T/C-values of the respective monotherapy.IC₅₀ and IC₇₀ values, being the drug concentration necessary to inhibitcolony formation by 50% (T/C=50%) and 70% (T/C=30%) respectively, weredetermined by plotting compound concentration versus relative colonycount.

The results are shown in FIG. 9. When the human mammary cancer xenograftMAXF 857 was treated with 10 μM roscovitine alone this gave a relativecolony count of 77%. When this cell line was treated with thecombination of roscovitine and 5FU the relative colony count wasapproximately 45% at concentrations of 5FU that had no effect on cellgrowth when treated alone. Thus the reduction from 77% to 45% suggestssignificant synergy in this concurrent treatment.

By way of summary, the efficacy of combination treatment usingroscovitine and 5-FU has been demonstrated using a variety ofexperimental methods, including cell cycle analysis, cell proliferationstudies, and studies on the induction of apoptosis. It is noteworthythat the results of studies using MCF7 cells indicate the presence of asynergistic effect where 5-FU and roscovitine are administeredconcurrently, and also for pretreatment with 5-FU. Studies on theinduction of apoptosis as measured by the activated caspase-3 assayindicate the presence of a synergistic effect for pretreatment withroscovitine.

Thus, the experimental results outlined above provide evidence ofsynergism where 5-FU and roscovitine are administered concurrently, andfor sequential administration in any order, i.e. where 5-FU isadministered prior to roscovitine, and where roscovitine is administeredprior to 5-FU.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the relevant fields are intended to be covered by the presentinvention.

1. A combination comprising a CDK inhibitor and 5-FU, or a prodrugthereof.
 2. A combination according to claim 1 wherein the CDK inhibitoris an inhibitor of CDK2 or CDK4.
 3. A combination according to claim 1or claim 2 wherein the CDK inhibitor is selected from rosovitine,purvalanol A, purvalanol B and olomoucine.
 4. A combination according toany preceding claim wherein the CDK inhibitor is roscovitine.
 5. Acombination according to any preceding claim wherein the prodrug of 5-FUis capecitabine.
 6. A pharmaceutical composition comprising acombination according to any preceding claim and a pharmaceuticallyacceptable carrier, diluent or excipient.
 7. Use of a combinationaccording to any one of claims 1 to 5 in the preparation of a medicamentfor the treatment of a proliferative disorder.
 8. Use according to claim7 wherein the proliferative disorder is cancer.
 9. Use according toclaim 8 wherein the cancer is breast cancer.
 10. A pharmaceuticalproduct comprising a CDK inhibitor and 5-FU, or a prodrug thereof, as acombined preparation for simultaneous, sequential or separate use intherapy.
 11. A pharmaceutical product according to claim 10 for separateor sequential use in therapy, wherein the 5-FU, or prodrug thereof, andCDK inhibitor are administered sequentially.
 12. A pharmaceuticalproduct according to claim 10 or claim 11 wherein the CDK inhibitor isan inhibitor of CDK2 or CDK4.
 13. A pharmaceutical product according toany one of claims 10 to 12 wherein the CDK inhibitor is selected fromrosovitine, purvalanol A, purvalanol B and olomoucine.
 14. Apharmaceutical product according to any one of claims 10 to 13 whereinthe CDK inhibitor is roscovitine.
 15. A pharmaceutical product accordingto any one of claims 10 to 14 wherein the prodrug of 5-FU iscapecitabine.
 16. A pharmaceutical product according to any one ofclaims 10 to 15 in the form of a pharmaceutical composition comprising apharmaceutically acceptable carrier, diluent or excipient.
 17. Apharmaceutical product according to any one of claims 10 to 16 for usein the treatment of a proliferative disorder.
 18. A pharmaceuticalproduct according to claim 17 wherein the proliferative disorder iscancer.
 19. A pharmaceutical product according to claim 18 wherein theproliferative disorder is breast cancer.
 20. A method of treating aproliferative disorder, said method comprising administering to asubject, simultaneously, sequentially or separately, 5-FU or a prodrugthereof, and a CDK inhibitor.
 21. A method according to claim 20 whichcomprises administering said CDK inhibitor to a subject prior tosequentially or separately administering 5-FU, or a prodrug thereof, tosaid subject.
 22. A method according to claim 20 which comprisesadministering 5-FU, or a prodrug thereof, to a subject prior tosequentially or separately administering a CDK inhibitor to saidsubject.
 23. A method according to any one of claims 20 to 22 whereinthe CDK inhibitor is an inhibitor of CDK2 or CDK4.
 24. A methodaccording to claim 23 wherein the CDK inhibitor is selected fromrosovitine, purvalanol A, purvalanol B and olomoucine.
 25. A methodaccording to claim 24 wherein the CDK inhibitor is roscovitine.
 26. Amethod according to any one of claims 20 to 25 wherein the prodrug of5-FU is capecitabine.
 27. A method according to any one of claims 20 to26 wherein the CDK inhibitor and 5-FU, or prodrug thereof, are eachadministered in a therapeutically effective amount with respect to theindividual components.
 28. A method according to any one of claims 20 to26 wherein the CDK inhibitor and 5-FU, or prodrug thereof, are eachadministered in a subtherapeutic amount with respect to the individualcomponents.
 29. A method according to any one of claims 20 to 28 whereinthe proliferative disorder is cancer.
 30. A method according to any oneof claims 20 to 29 wherein the proliferative disorder is breast cancer.31. Use of a CDK inhibitor in the preparation of a medicament for thetreatment of a proliferative disorder, wherein said treatment comprisesadministering to a subject simultaneously, sequentially or separately5-FU, or a prodrug thereof, and a CDK inhibitor.
 32. Use of a CDKinhibitor and 5-FU, or a prodrug thereof, in the preparation of amedicament for treating a proliferative disorder.
 33. Use of a CDKinhibitor in the preparation of a medicament for the treatment of aproliferative disorder, wherein said medicament is for use incombination therapy with 5-FU, or a prodrug thereof.
 34. Use of 5-FU, ora prodrug thereof, in the preparation of a medicament for the treatmentof a proliferative disorder, wherein said medicament is for use incombination therapy with a CDK inhibitor.